Novel lymphokine, monoclonal antibody specific to the lymphokine, and their production and uses

ABSTRACT

A monoclonal antibody specific to the lymphokine, and its production is disclosed. The novel lymphokine LK2 is a glycoprotein with a molecular weight of 20,000±2,000 daltons; isoelectric point pI, 6.2±0.3; electrophoretic mobility Rf, 0.29±0.02; cytotoxic on L 929 cells and substantially not growth-inhibitive on KB cell. The lymphokine significantly inhibits the growth of malignant human tumors in vivo. The monoclonal antibody may be of IgM or IgG class, and neutralizes specifically the cytotoxic activity of the lymphokine. Combined use of LK 2 with chemotherapeutic agents such as alkylating agents, metabolic antagonists, antioncotic antibiotics and plant alkaloids enhances greatly the antioncotic effect of the chemotherapeutics.

this is a division of application Ser. No. 792,158 filed Oct. 28, 1985.

FIELD OF THE INVENTION

The present invention relates to a monoclonal antibody specific to anovel lymphokine, and the production thereof.

BACKGROUND OF THE INVENTION

Lymphotoxin (LT) and tumor necrosis factor (TNF) are known aslymphokines which damage tumor cells. For example, LT is described inAoki, Ryuichi et al., SHIN-MENEKIGAKU SOSHO, Vol. 6, "Lymphokine", pp.87-105 (1979), published by Igaku-Shoin, Tokyo, In Vitro Method inCell-Mediated Immunity, edited by Bloom, B. R. & Glade, P. R., publishedby Academic Press, Inc. (1971), and Cellular Immunology, Vol. 38, pp.388-402 (1978); and TNF is described in Carswell, E. A. et al.,Proceedings of the National Academy of Sciences of the U.S.A., Vol. 72,No. 9, pp. 3,666-3,670 (1975), and Lymphokines, Vol. 2, pp. 235-272,"Tumor Necrosis Factor", edited by Pick, E., published by AcademicPress, Inc. (1981).

Recently, Ohnishi, H. et al. disclosed an antioncotic lymphokineglycoprotein in Japan Patent Kokai No. 146,293/83.

Milstein, C. reviewed monoclonal antibodies in Scientific American, Vol.243, No. 4, pp. 56-64 (1980).

In chemotherapeutics, generally one or more alkylating agents, metabolicantagonists, antioncotic antibiotics, and plant alkaloids are used.

Chemotherapeutics, however, have the disadvantages that their use maycause excessive side-effects in patients; that their tumor spectra arerelatively narrow and insufficient; and that they are liable to inducedrug-resistant tumors.

DETAILED DESCRIPTION OF THE INVENTION

In view of the foregoing, the present inventors have studied lymphokinesover a period of years. As the result, they have discovered a novellymphokine with physicochemical properties entirely different from thoseof known lymphokines, and having cytotoxic activity on malignant tumorcells. Thus, they have established the production and uses of thelymphokine, in addition to the monoclonal antibody specific to thelymphokine and its production.

More particularly, the present invention relates to a novel lymphokinepossessing the following physicochemical properties:

(1) Molecular weight: 20,000±2,000;

(2) Isoelectric point: pI=6.2±0.1;

(3) Electrophoretic mobility: on Disc-PAGE, Rf=0.29±0.02;

(4) uv-Absorption spectrum: an absorption maximum at a wave length ofabout 280 nm;

(5) Solubility in solvents:; soluble in water, saline and phosphatebuffer, scarcely soluble or insoluble in ethyl ether, ethyl acetate orchloroform;

(6) Coloring reaction protein-positive by Lowry's method or themicroburet method, saccharide-positive by the phenol-sulfuric acidmethod or anthrone-sulfuric acid method;

(7) Biological activities: cytotoxic on L 929 cell, substantially notgrowth-inhibitive on KB cell substantially free of interferon activity;

(8) Stability in aqueous solution: stable up to 60° C. when incubated atpH 7.2 for 30 minutes, stable in the pH range of 4.0-11.0 when incubatedat 4° C. for 16 hours; and

(9) Stability on cryopreservation: stable at -10° C. over a period ofone month or longer.

The present invention also relates to the production and uses of thelymphokine, as well as to the monoclonal antibody specific to thelymphokine and production thereof.

The novel lymphokine in accordance with the present invention will beabbreviated simply as "LK 2" hereinafter.

LK 2 is produced by exposing an LK 2 producing human cell, e.g., humanleukocytes, human lymphocytes or established cell lines thereof, to anLK 2 inducer. Human leukocytes lymphocyte can be isolated from freshhuman blood. The established human cell line can be proliferated withconventional in vitro methods.

For a more efficient practice of the present invention, it is desirableto employ an in vivo cell proliferation procedure wherein the human cellline is transplanted directly in to a non-human warm-blooded animal, or,alternatively, inoculated in a conventional-type diffusion chamber bywhich the nutrient body fluid of a non-human warm-blooded animal issupplied to the cell line.

Unlike in vitro cell proliferation, the in vivo procedure requires no ormuch less nutrient medium containing expensive serum, the cellproliferation is much easier, and the proliferated human cells give amuch higher LK 2 activity.

In addition, in the in vivo procedure the human cell line can be easilyproliferated while utilizing the nutrient body fluid supplied from anon-human warm-blooded animal by transplanting the human cell line tothe non-human warm-blooded animal, or, alternatively, placing the cellline in a conventional-type diffusion chamber devised to receive thebody fluid, and embedding or placing the chamber in or on the animal. Ineither case, the animals are fed in the usual way.

Furthermore, the in vivo procedure has the additional features of a muchmore stabilized and rapid cell proliferation, a higher cell production,and an extremely higher LK 2 production per cell, as compared with thein vitro procedure.

The human cell lines usable in the present invention may be those whichare LK 2-producible, transplantable to a non-human warm-blooded animal,and readily proliferatable in the animal. For example, a variety ofhuman cell lines listed in Protein, Nucleic Acid and Enzyme, Vol. 20,No. 6, pp. 616-643 (1975) are employable in the invention. Specificallysuited are human lymphoblastoid lines, such as Namalwa (ATCC CRL 1432),as described in Journal of Clinical Microbiology, Vol. 1, pp. 116-117(1975); BALL-1, TALL-1 and NALL-1, as described by Miyoshi, I., Nature,Vol. 267, pp. 843-844 (1977); M-7002 and B-7101, as described in TheJournal of Immunology, Vol. 113, pp. 1,334-1,345 (1974); JBL, EBV-Sa,EBV-Wa, MOLT-3 (ATCC CRL 1552) and EBV-HO, as described in The TissueCulture, Vol. 6, No. 13, pp. 527-546 (1980); CCRF-SB (ATCC CCL 120);CCRF-CEM (ATCC CCL 119); BALM 2; DND-41; and other established celllines obtained by transforming normal human monocyte or granulocytecells with any carcinogenic virus, agent or radiation.

The proliferation rate and/or LK 2 productivity per cell of these celllines may be improved by cell fusion techniques using polyethyleneglycol or Sendai virus, or by gene recombinant techiques using nucleaseenzyme, ligase enzyme, DNA polymerase enzyme, etc. The listing of theemployable human cell lines in the present specification is not intendedin any way to limit the scope of the invention. One or more members ofthese cell lines may be used in combination in the steps up to the LK 2induction which will be described hereinafter. If necessary, humanleakocytes or lymphocyte, which can be prepared from fresh human blood,may be used in combination with any of the human cell lines.

The non-human warm-blooded animal usable in the invention may be any oneof those wherein in which such human cells are proliferatable. Examplesof such animals are fowl, such as chicken and pigeon; and mammals, suchas dog, cat, monkey, rabbit, goat, pig, horse, cow, guinea pig, rat,nude rat, hamster, mouse, nude mouse, etc.

Since transplantation of the human cells to the animal can elicit anundesirable immunoreaction, the use of a non-human warm-blooded animalin the youngest possible stage, e.g., egg, embryo or foetus, or newbornor infant animal, is desirable in order to reduce such immunoreaction asfar as possible.

Prior to the transplantation, the animal may be irradiated with x-ray orγ-ray, about 200-600 rem, or injected with a antiserum or animmunosuppressant to reduce the immunoreaction to the lowest possiblelevel.

When an immunodeficient animal, such as nude mouse and nude rat, is usedas the host animal, any of the aforementioned human cell lines can betransplanted into these animals without such pretreatment, andproliferated readily with less fear of causing undesirableimmunoreaction because these animals exhibit less immunoreaction even intheir adulthood.

One can stabilize cell proliferation and/or augment LK 2 production bysuccessive transplantation using the same or different non-humanwarm-blooded animals. These objectives can be attained, for example, byfirst transplanting a human cell line to a hamster and proliferating thehuman cell line in the hamster, then successively transplanting theproliferated human cell to a nude mouse. In this case, the successivetransplantation may be carried out with a non-human warm-blooded animalof the same class or order, as well as those of the same species orgenus.

The human cells can be transplanted into any site of the animal as longas the human cell proliferates in that site: for example, in theallantoic cavity, or intravenously, intraperitoneally, orsubcutaneously.

Alternatively, the human cells may be proliferated by placing them in aconventional diffusion chamber of an of various shapes and sizes,equipped with a suitable means which prevents contamination of thechamber carring the animal cells, but supplies the human cells with thenutrient body fluid of the animal, e.g., a membrane filter, ultrafilteror hollow fiber of a nominal pore size of about 10⁻⁷ -10⁻⁵ m; embedding,for example, intraperitoneally, the chamber in the animal; and allowingthe human cells to proliferate in the chamber while receiving thenutrient body fluid from the animal.

Furthermore, the diffusion chamber can be designed and placed, e.g. onthe animal, so that the nutrient fluid in the chamber can circulatefreely through the chamber. The culture in the chamber can be observedduring the cell proliferation through transparent side window(s),equipped on the chamber wall(s), and/or the chamber per se can bereplaced at intervals with a fresh one, both to continue the cellproliferation over the period of the life span of the animal withoutsacrificing and to augment much more the cell production per animal.Since due to the absence of direct contact of the human cells with theanimal cells, such a diffusion chamber technique elicits much lessundesirable immunoreaction. Thus, any non-human warm-blooded animal maybe readily used, without pretreatment to reduce such immunoreaction, andthe proliferated viable human cells can be harvested easily from thediffusion chamber.

Feeding of the animal can be carried out in the usual way, and nospecial care is required even after the transplantation. The periodrequired to obtain maximum cell proliferation is generally from one to10 weeks. The number of the human cells so obtained is about 10⁷ -10¹²cells per animal or more. More particularly, according to the presentinvention, the transplanted human cells increase by to about 10² -10⁷-fold or more, which is about 10-10⁶ -fold or higher than that obtainedby inoculating and proliferating the human cells on an in vitro nutrientculture medium. This is very favorable in the production of LK 2.

Any method is employable in the present invention as long as LK 2production can be induced in the proliferated human cells therewith. Theproliferated human cells may be exposed to an LK 2 inducer in the animalused as the host for cell proliferation. For example, human cells,proliferated in ascite in suspension, or tumor cells, formed, e.g.,subcutaneously, may be directly exposed in vivo to an LK 2 inducer toinduce LK 2 production, and the accumulated LK 2 harvested from theascite, serum and/or tumor, followed by purification of the LK 2.Alternatively, the proliferated human cells may be harvested from theanimal and then exposed in vitro to an LK 2 inducer. For example, theproliferated human cells, obtained by harvesting from ascite suspension,or extracting and disaggregating the tumor mass(es), formed, e.g.,subcutaneously, may be suspended in a nutrient culture medium, prewarmedto a temperature of about 20°-40° C., to give a cell density of about10⁵ -10⁸ cells/ml, and exposed in vitro to an LK 2 inducer, followed bythe accumulated LK 2 from the culture.

When a conventional-type diffusion chamber is used, exposure of theproliferated human cells to an LK 2 inducer is carried out in thechamber or after harvesting.

The human cells so obtained may be cultured in vitro for an additional1-4 days to regulate their generation time, prior to the LK 2 induction.

The LK 2 production per animal may be further augmented by employing oneor more of the following methods:

(1) a method wherein the proliferated human cells are exposed to an LK 2inducer in the animal, which has been used as the host for the cellproliferation, and then harvested from certain site(s) of the animal orits whole body, followed by in vitro exposure of the human cells to anLK 2 inducer,

(2) a method wherein the human cells are repeatedly exposed to an LK 2inducer, and

(3) a method wherein the diffusion chamber embedded in or connected tothe animal is replaced at intervals with a fresh one.

The LK 2 inducers usable in the present invention are conventionalα-interferon inducers (IFN-α inducers), such as virus, nucleic acid andnucleotide; and conventional γ-interferon inducers (IFN-γ inducers),such as, phytohaemagglutinin, concanavalin A, pokeweed mitogen,lipopolysaccharide, endotoxin, polysaccharide and bacteria. Antigens acton sensitized cells as an LK 2 inducer.

The LK 2 production can be augmented by a combined use of IFN-α andIFN-γ inducers as LK 2 inducer. It has been confirmed that suchcombination induces simultaneous production of human-specific interferon(HuIFN). This is very advantageous in a simultaneous and low-costmass-production of two or more biologically-active substances, i.e.,invaluable LK 2 and HuIFN, as well as in a much more effectiveutilization of human cells.

The LK 2 so obtained can be recovered by one or more purification and/orseparation procedures, e.g., salting-out, dialysis, filtration,centrifugation, concentration, and/or lyophilization. If a much morepurified LK 2 preparation is desirable, a preparation of the highestpurity can be obtained by the above described procedure(s) incombination with other conventional procedure(s), e.g., adsorption anddesorption with ion exchange, gel filtration, isoelectric pointfractionation, electrophoresis, ion exchange chromatography,high-performance liquid chromatography, column chromatography, and/oraffinity chromatography.

Immobilized monoclonal antibodies obtained by binding a monoclonalanti-LK 2 antibody, which will be described hereinafter, onto a suitablewater-insoluble carrier, e.g., BrCN-activated Sepharose, a product ofPharmacia Fine Chemical AB, Uppsala, Sweden, can be advantageously usedto speed up and facilitate purification of LK 2.

It was confirmed that LK 2 thus obtained has the followingphysicochemical properties:

(1) Molecular weight: 20,000±2,000 daltons;

(2) Isoelectric point: pI=6.2±0.3;

(3) Electrophoretic mobility: on Disc-PAGE, Rf=0.29±0.02;

(4) uv-Absorption spectrum: an absorption maximum at a wave length ofabout 280 nm:

(5) Solubility in solvents: soluble in water, saline and phosphatebuffer, scarcely soluble or insoluble in ethyl ether, ethyl acetate orchloroform;

(6) Coloring reaction: protein-positive by Lowry's method or themicroburet method, saccharide-positive by the phenol-sulfuric acidmethod or anthrone-sulfuric acid method;

(7) Biological activities: cytotoxic on L 929 cells, cytostatic on KBcell substantially free from interferon activity;

(8) Stability in aqueous solution: stable up to 60° C. when incubated atpH 7.2 for 30 minutes, stable in the pH range of 4.0-11.0 when incubatedat 4° C. for 16 hours; and

(9) Stability on cryopreservation: stable at -10° C. over a period ofone month or longer.

It has also been confirmed that LK 2 does not effect any substantialcytolysis on normal human cells, but effects a remarkable cytolysis on avariety of human tumor cells as well as on the mouse fibroblastoid line,L 929, to kill these cells. Thus, LK 2, e.g., in the form of acomposition, is suitable use against prophylactic and/or therapeutic forLK 2-sensitive diseases, e.g., malignant tumors, and more particularly,against various malignant human tumors, the treatment of which haspreviously been deemed very difficult.

According to one aspect of the present invention, the antioncoticeffects of conventional chemotherapeutics can be enhanced with anenhancer that contains LK 2 as the effective component.

The dose of chemotherapeutics can be reduced to about 178-1/1000, of theconventional dos because the antioncotic effect of chemotherapeutics canbe greatly enhanced with the enhancer of the present invention. Inaddition, the use of the enhancer broadens the tumor spectra ofchemotherapeutics, and enables the treatment of drug-resistant tumors.

The chemotherapeutics usable in the invention include, for example oneor more alkylating agents, such as melphalan, cyclophosphamide,ifosfamide, estramustine sodium phosphate, busulfan, imorposulfantosilate, N-methyl-3,3'-dimesyloxydipropylaminebiphenyl-4,4'-disulfonate, carboquone, thio-TEPA, carmustine, nimustinehydrochloride, streptozocin, dacarbazine, and pipobroman; metabolicantagonists, such as methotrexate, fluorouracil, tegaful, carmoful,cytarabine, ancitabine hydrochloride, enocitabine, 6-mercaptopurine, andthioinosine; antioncotic, antibiotics such as doxorubicin, daunorubicin,aclarubicin, bleomycin, peplomycin, mitomycin C, actinomycins D and C,chromomycin A₃, mithramycin, and neocarzinostatin; and plant alkaloids,such as vincristine sulfate, vinblastine sulfate, vindesine sulfate, andpodophyllotoxin. In the case of treating prostate cancer or breastcancer, one or more antioncotic hormones, such as predonisolone,methyltestosterone and conjugated estrogen, can be desirably used incombination.

Conditions for combining these chemotherapeutics and LK 2 should bedesirably chosen, and not restricted.

The process for producing a monoclonal antibody according to the presentinvention comprises immunizing a non-human warm-blooded animal using LK2 as the antigen; isolating the antibody-producing cells from the bodyof the animal; fusing the antibody-producing cells with myeloma cells;selecting from the resultant hybrid cells a clone capable of producingan antibody which is specific to LK 2; proliferating the clone; andallowing the proliferated cell to produce the monoclonal antibodyspecific to LK 2.

Such immunization can be obtained by injecting, e.g., intravenously,intraperitoneally or subcutaneously, an aqueous solution, emulsion orsuspension of LK 2 as the antigen into a suitable non-human warm-bloodedanimal, e.g., chicken, pigeon, dog, cat, monkey, goat, pig, cow, horse,rabbit, guinea pig, rat, hamster or mouse; and feeding the animal forthree days or longer to induce antibody production. A conjugate of LK 2and a saccharide obtainable according to the teaching of Japan patentpublication No. 23,847/83 is also employable as the antigen.

The antigen may be injected in single dosage or, if necessary, in two ormore dosages at interval(s) of about 3-30 days.

The spleen cells of the immunized animal in which antibody productionhas been induced are fused with myeloma cells of the same or a differentspecies with a suitable procedure, e.g., those reported by Kohler, G. etal. in Nature, Vol. 256, pp. 495-497 (1975), and European Journal ofImmunology, Vol. 6, pp. 511-519 (1976). The hybrid cells so obtained arethen selected and cloned, after which the clone(s) is cultured in vitroor in vivo, followed by recovery of the accumulated highly-specificmonoclonal antibody from the resultant culture. Specifically, an in vivoprocedure is more preferable than the in vitro procedure because theformer procedure attains a much higher proliferation of the clone and amuch higher production of the monoclonal antibody without usingexpensive serum.

In the in vivo procedure, the clone is proliferated while utilizing thenutrient body fluid of a non-human warm-blooded animal of the same or adifferent species than that used in the immunization by transplantingthe clone to such a non-human warm-blooded animal, or inoculating theclone in a conventional-type diffusion chamber devised to receive thenutrient body fluid of such an animal, and the accumulated monoclonalantibody is recovered from the body fluids, such as ascite and serum.Alternatively, after proliferating in vivo, the clone may be cultured ona serum-free culture medium for an additional 1-5 days, followed byrecovery of the accumulated monoclonal antibody from the resultantculture.

The monoclonal antibody so obtained can be recovered easily with one ormore separation and/or purification procedures, e.g., salting-out,dialysis, filtration, centrifugation, concentration and/orlyophilization. If a much higher purification is desirable, apreparation of the highest purity can be obtained by the above-mentionedprocedure(s) in combination with other conventional procedure(s), e.g.,adsorption and desorption with ion exchange, gel filtration, isoelectricpoint fractionation, electrophoresis, ion exchange chromatography,high-performance liquid chromatography, column chromatography, and/oraffinity chromatography. The recovery yield of the monoclonal antibodycan be improved advantageously by use of an immobilized LK 2 gelobtained by binding a high-purity LK 2 onto a suitable water-insolublecarrier, e.g., BrCN-activated Sepharose.

The monoclonal antibody obtained according to the present invention isfavorably usable as a ligand for affinity chromatography directed to LK2 production, as well as in diagnosis of a variety of human diseasesbecause of its specificity to LK 2 which damages malignant tumors.

LK 2 titers are assayed by use of either KB cell or L 929 cell as thetarget cell: When KB cell is used, the cytostatic activity on KB cell isdetermined according to the method described in Cancer ChemotherapyReports Part 3, Vol. 3, No. 2, Sept. (1972); When L 929 cell is used,the cytotoxic activity on L 929 cell in the presence of actinomycin Dwas determined by the method described in Lymphokines, Vol. 2, pp.245-249, "Tumor Necrosis Factor", edited by Pick, E., published byAcademic Press Inc. (1981). One unit is defined as the amount of LK 2that kills 50% of L 929 cells when cultured in the presence ofactinomycin D for 18 hours. Throughout the present specification, thelatter method using L 929 cell was employed unless otherwise specified.

The titers of HuIFN were assayed by the conventional plaque-reductionmethod using FL cells of human amnion origin described in Protein,Nucleic Acid and Enzyme, Vol. 20, No. 6, pp. 616-643 (1975).

The haemagglutination titers were assayed according to the methodreported by Salk, S.E., The Journal of Immunology, Vol. 49, pp. 87-98(1944).

The following Experiments further detail the present invention.

EXPERIMENT A-1 Preparation of Partially-Purified LK 2

Newborn hamsters were injected with about 4 mg/hamster of conventionalantiserum prepared from rabbit to weaken their possible immunoreactionupon heterotransplantation, transplanted subcutaneously with BALL-1cells, about 1×10⁷ cells/hamster of cell, a human lymphoblastoid line,and fed in the usual way for three weeks. The tumor masses, formedsubcutaneously, were extracted, minced, and disaggregated in saline. Tothe cell suspension so obtained was then washed RPMI 1640 medium (pH7.2) supplemented with serum, and resuspended in a fresh preparation ofthe same culture medium to give a cell density of about 2×10⁶ cells/ml.The cell suspension was added with Sendai virus (about 400haemagglutination titers/ml), and incubated at 37° C. for 24 hours toinduce LK 2 production.

The culture was centrifuged at about 1,000×g and about 4° C., and theresultant precipitate was removed. The supernatant so obtained wasdialyzed against saline containing 0.01M phosphate buffer (pH 7.2) for20 hours, and treated with a membrane filter. The filtrate was thenpassed through a column of "NK-2", an immobilized monoclonalanti-HuIFN-alpha antibody commercialized by CELLTECH, Burkshire, UK, andthe unadsorbed fraction was collected. An active fraction was recoveredfrom this fraction by means of chromatofocusing, concentrated, andlyophilized to obtain a pulverulent product with LK 2 activity.

The specific activity of the product was about 10⁶ units/mg protein. TheLK 2 yield was about 2.0×10⁷ units per hamster.

EXPERIMENT A-2 Preparation of Anti-LK 2 Antibody

An LK 2 preparation, obtained by the method in Experiment A-1, wasdissolved in saline to give a concentration of about 0.05 w/v % asprotein, and to the solution was added the same volume of Freund'scomplete adjuvant. Mice were immunized by subcutaneously injecting 0.2ml aliquots of the mixture so obtained, and boosting seven days afterthe first injection. After inducing anti-LK 2 antibody production in theantibody-producing cells of the animals, the spleens of the animals wereextracted, minced, disaggregated, and suspended together with a mousemyeloma cell line, P₃ -X63-Ag8 (ATCCT1B9), purchased from FlowLaboratories Inc., Rockville, Md., USA, in serum-free Eagle's minimalessential medium (pH 7.2) containing 50 w/v % polyethylene glycol 1000,prewarmed to 37° C., to give a respective cell density of 10⁴ cells/ml,followed by a 5-minute standing of the resultant mixture. Thereafter,the mixture was diluted 20-times in a fresh preparation of the sameculture medium, and the hybridoma cells capable of growing on thehypoxanthine, aminopterin, thymidine containing medium were collectedaccording to the method reported by Davidson, R. L. and Gerald, P. S. inSomatic Cell Genetics, Vol. 2, pp. 175-176 (1976) to clone the hybridomacells capable of producing anti-LK 2 antibody. Mice were transplantedintraperitoneally with the cloned hybridoma cells in a dosage of about10⁶ cells per mouse, fed for two weeks, and sacrificed. The body fluidsof the animals, such as ascite fluid and blood, were recovered,centrifuged, and salted out with ammonium sulfate, followed bycollection of the fractions sedimented at 30-50% saturation. Thesefractions were dialyzed, and subjected to affinity-chromatography usingan immobilized anti-(LK 2 antibody) gel obtained by reacting an LK 2specimen, prepared by the method in Experiment A-1, with BrCN-activatedSepharose at ambient temperature, to obtain an anti-(LK 2 antibody)fraction which was then dialyzed, concentrated and lyophilized.

The resultant pulverulent product exhibited an immunologically-specificneutralization to the cytotoxic activity of LK 2.

The stability of the monoclonal antibody in aqueous solution was studiedby assaying the residual neutralizing activities after incubating underprescribed conditions: On incubation at pH 7.2 and differenttemperatures for 30 minutes, 80% or more of the activity was retained at60° C., but 90% or more was lost at 70° C. After incubation at 4° C. anddifferent pH levels for 16 hours, the activity was stable in the pHrange of 4.0-11.0, but was lost by 90% or more at pH 2.0.

On studying the properties of the monoclonal antibody, the monoclonalantibody was found not to be resistant to 2-mercaptoethanol, andeffected a specific antigen-antibody reaction with anti-mouseimmunoglobulin M antibody. Thus, the present monoclonal antibody isgrouped into the class of immunoglobulin M antibody.

EXPERIMENT A-3 Prepartation and Physicochemical Properties ofHighly-purfied LK 2

A partially-purified LK 2 specimen, obtained by the method in ExperimentA-1, was subjected to affinity-chromatography using an immobilized gelof the monoclonal antibody prepared by the method in Experiment A-2, tocollect LK 2 fractions which were then dialyzed, concentrated andlyophilized.

The resultant was a highly-purified LK 2 preparation with a specificactivity of about 10⁹ units/mg protein.

The physicochemical properties of LK 2 were studied with thispreparation.

(1) Molecular weight:

The molecular weight of LK 2 was determined by the electrophoreticmethods using SDS-polyacrylamide gel described in Weber, K. and Osborn,M., Journal of Biological Chemistry, Vol. 244, page 4,406 (1969).Columns of 10% acrylamide gel were loaded with about 10 μg aliquots ofthe preparation in the presence of 0.1% SDS, and charged with 8 mA percolumn for four hours to effect electrophoresis. After extraction andsubsequent LK 2 assay of the active fractions, the molecular weight ofLK 2 was found to be 20,000±2,000 daltons.

(2) Isoelectric point:

A 2 hour, 25 W electrofocusing of the preparation using "AMPHOLINEPAGPLATE (pH 3.5-9.5)", a gel product for electrofocusing,commercialized by LKB-Produkter AB, Stockholm, Sweden, gave anisoelectric point pI of 6.2±0.3.

(3) Electrophoretic mobility:

According to the method described in Davis, B. J., Annals of New YorkAcademy of Sciences, Vol.121, page 404 (1964), about 10 μg aliquots ofthe preparation were loaded on columns of 7.5% acrylamide gel, subjectedto electrophoresis at pH 8.3 and 3 mA per column for two hours,extracted, and assayed for LK 2 activity to obtain an electrophoreticmobility Rf of 0.29±0.02.

(4) uv-Absorption spectrum:

After analyzing the uv-spectrum of the preparation with UV-250spectrometer, a product of Shimadzu Seisakusho KK, Kyoto, Japan, anabsorption maximum was found at a wave length of about 280 nm.

(5) Solubility in solvent:

Soluble in water, saline and phosphate buffer solution; scarcely solubleor insoluble in ethyl ether, ethyl acetate and chloroform.

(6) Coloring reaction:

Protein-positive by Lowry's method and the microburet method;saccharide-positive by the phenol-sulfuric acid method and theanthrone-sulfuric acid method.

(7) Biological activity:

A cytotoxic activity on L929 cell and no substantial growth-inhibitoryactivity on KB cell were noted. No substantial HuIFN activity was noted.

(8) Stability in an aqueous solution:

(i) Heat stability:

About 1×10⁵ units/ml aliquots of the preparation were incubated at pH7.2 and different temperatures for 30 minutes, and the residualcytotoxic activities were assayed. As a result, LK 2 was found stable upto 60° C.

(ii) pH Stability:

0.1 ml aliquots of the preparation (1×10⁶ units/ml) were combined with 1ml buffer solution of different pH levels, i.e. McIlvaine buffer at pH2-7; phosphate buffer, pH 7-8; glycine-NaOH buffer, pH 8-11, andincubated at 4° C. for 16 hours. Thereafter, 0.1 ml of the incubatedmixture was adjusted to pH 7.2 with 0.05M phosphate buffer (pH 7.2), andthe residual activity was assayed. As a result, LK 2 was found stable inthe pH range of 4.0-11.0.

(iii) Stability to "DISPASE":

To about 1×10⁵ units/per ml of the preparation was added "DISPASE", aprotease enzyme of Bacillus microorganism, commercialized by Godo ShuseiCo., Ltd., Tokyo, Japan, to give an enzyme activity of 100 units/ml, andthe mixture was incubated at pH 7.2 and 37° C. for two hours. During theincubation, small portions of the mixture were sampled periodically, andcombined with calf serum albumin to give a concentration of 1 w/v % tosuspend the enzymatic reaction. On assaying the LK 2 activities in thesamples, LK 2 was susceptive to DISPASE treatment and lost its activityas the enzymatic reaction proceeded.

(9) Stability to cryopreservation:

The LK 2 preparation was stored in aqueous solution at -10° C. and pH7.2 for one month, thawed and assayed. No decrease in activity wasnoted.

From these evidences, it is clear that LK 2 has physicochemicalproperties distinguishable from those of known lymphokines such as LT,TNF or IFN. Also, the present monoclonal antibody is novel because itexhibits an immunologically-specific neutralization with the cytotoxicactivity of the novel lymphokine LK 2.

EXPERIMENT B-1 Cytostatic Effect on Malignant Tumor Cells

The cytostatic activity of LK 2 on several human cells was studied withLK 2 preparations obtained by the method in Experiments A-1 and A-3.

One human cell suspension (10⁶ cells) listed in Table I was suspended in1 ml of conventional nutrient medium supplemented with foetal calfserum, cultured for one day, added with 0.1 ml of a saline containingeither 50 units or 500 units of an LK 2 preparation, prepared by themethod in Experiment A-1 or A-3, and incubated at 37° C. for two days.After completion of the culture, the viable cells were stained withneutral red, a type of staining agent, according to the method describedin Applied Microbiology, Vol. 22, No. 4, pp. 671-677 (1971), and thestaining agent was eluted by use of an acidified ethanol solution. Thenumber of the viable cells was determined by measuring the absorbance ofthe eluate at a wave length of 540 nm.

                                      TABLE I                                     __________________________________________________________________________                       LK 2 at Experiment A-1                                                                    LK 2 at Experiment A-3                         Name of cell line                                                                      Source of cell line                                                                     50 units                                                                            500 units                                                                           50 units                                                                            500 units                                __________________________________________________________________________    Hep #2*  Larynx epidermoid                                                                       33    40    46    68                                                carcinoma                                                            PC-8*    Lung carcinoma                                                                          26    37    56    80                                       MKN 7*   Gastric cancer                                                                          35    41    60    77                                       HLE*     Liver carcinoma                                                                         32    38    55    71                                       HeLa*    Cervix epitheloid                                                                       24    35    46    68                                                carcinoma                                                            L-132**  Embryonic lung                                                                           3    -2     1    -3                                       Chang liver**                                                                          Liver      2    -3    -4    -1                                       Giradi heart**                                                                         Heart     -2     2    -1    -2                                       __________________________________________________________________________     Note: *indicates human cell lines of malignant tumor origins; **those of      normal origins.                                                          

As a control, 0.1 ml of an LK 2 free saline was used.

Growth inhibition (%) was calculated with the following equation:##EQU1##

The results are given in Table I.

These results confirm that LK 2 does not substantially affect normalcells, but greatly inhibits the growth of various malignant tumor cells.It was also confirmed that the effect of a partially-purified LK 2compares well with that of highly-purified one.

EXPERIMENT B-2

A group of ten BALB/c mice was transplanted with Mrth A cell of mousesarcoma origin. From the tenth day after the transplantation, the micewere injected intravenously with saline containing an LK 2 preparation,obtained by the method in Experiment A-3, in a dosage of 100 or 1,000units/kg daily for 15 days. Thereafter, the mice were sacrificed, andthe tumor masses, formed in the animals, were measured.

The results are shown in Table II.

                  TABLE II                                                        ______________________________________                                                     Dosage per day                                                                            Tumor mass                                           Treatment    (units/kg)  (g)                                                  ______________________________________                                        Control       0          5.7 ± 0.7                                         LK 2         100         3.3 ± 0.4*                                                     1,000       2.8 ± 0.4*                                        ______________________________________                                         Note: *means the values are statistically significant against the control     in a level of significance of 5%.                                        

EXPERIMENT B-3

A group of BALB/c nude mice was transplanted subcutaneously in theirdorsum areas with small fragments of human breast cancer tissue.

After the tumor masses grew to about 200 mm³ in the bodies of theanimals, saline containing an LK 2 preparation, obtained by the methodin Experiment A-1 or A-3, was injected intravenously once every day in adosage of either 100 units/kg or 1,000 units/kg for twenty days.Thereafter, the animals were sacrificed, and the resultant tumor masseswere weighed.

The results are given in Table III. T1 TABLE III-? Dosage per day? Tumormass? -Treatment? (units/kg)? (g)? -Control 0 10.8 ± 1.0 -LK 2 100 7.3 ±0.7* -at Experiment A-1 1,000 6.8 ± 0.5* -LK 2 100 6.4 ± 0.5* -atExperiment A-3 1,000 5.6 ± 0.7* -

EXPERIMENT B-4

A group of BALB/c nude mice was transplanted subcutaneously in theirdorsum areas with small fragments of human breast cancer tissuesimilarly as in Experiment B-3.

After the tumor masses grew to about 200 mm³ in the bodies of theanimals, saline containing an LK 2 preparation, obtained by the methodin Experiment A-3, and/or an HuIFN-α preparation, was injectedintravenously once every day for twenty days. Thereafter, the animalswere sacrificed, and the resultant tumor masses were weighed.

As the control, an LK 2- and HuIFN-α-free saline was used.

The results are given in Table IV.

These evidences clearly show that the combined use of LK 2 and HuIFN-αextremely enhances the antioncotic effect of HuIFN-α.

                  TABLE IV                                                        ______________________________________                                                      Dosage per day                                                                            Tumor mass                                          Treatment     (units/kg)  (g)                                                 ______________________________________                                        Control        0          10.8 ± 1.0                                       LK 2          100         6.4 ± 0.5*                                       HuIFN-α 1,000       6.7 ± 0.5*                                       LK 2          100         5.3 ± 0.4*                                       plus HuIFN-α                                                                          1,000                                                           ______________________________________                                         Note: *means the values were statistically significant against the contro     in a level of significance of 5%.                                        

EXPERIMENT B-5

An acute toxicity test, wherein a group of 20-day old mice wasadministrated with an LK 2 preparation, obtained by the method inExperiment A-3, confirmed that the toxicity of the preparation wasextremely low, i.e., an LD₅₀, of 10⁹ units or more, upon intraperitonealinjection.

As is obvious from the above experiments, LK 2 exhibits a strongcytostatic effect on malignant tumors in vitro as well as in vitro aswell as in vivo. Furthermore, the administration of LK 2 is very safe asa high dosage does not practically affect normal cells, while a lowdosage remarkably affects tumor cells.

The effective dosage of LK 2 generally falls in the range of5-500,000,000 units/day for an adult: more particularly, for localadministration, e.g., in the form of local injection or collyrium,5-10,000,000 units/day; for percutaneous or permucosal administration,e.g., in the form of ointment or suppository, 10-50,000,000 units/day;for systemic administration, e.g. intravenous- or intramuscularinjection, 50-100,000,000 units/day; and oral administration,500-500,000,000 units/day, but the dosage is freely variable dependentupon the instructions and the patient's symptoms.

Although LK 2 can be prepared into medicine in the usual way afteradmixing with a suitable conventional carrier, base and/or vehicle, theLK 2 content thereof should be at least 5 units/g in view of itstoxicity, effective dosage, and safety.

The shape and form of prophylactic- and/or therapeutic agents for LK2-sensitive diseases can be freely chosen: for example, for oraladministration, it may be formulated into preparations for enteric uses,e.g., capsule, tablet or powder; for rectal administration, suppository;for injection, it may be, for example, prepared into a lyophilizedinjection which is dissolved, prior to use, into an injection solutionwith distilled water, as well as in the forms of collunarium, collyriumor ointment.

As an example of the treatment of a malignant tumor patient, a tumortissue fragment extracted from the patient may be treated in vitro withLK 2 to enhance the immunogenicity of the tissue fragment, and thattreated tissue fragment, and administered to the patient to obtain amuch more effective treatment of the malignant tumor.

Combined uses of LK 2 with antioncotic(s), for example, lymphokines,such as HuIFN, TNF, LT and T-cell growth factor (TCGF); antioncoticpolysaccharides, such as β-1,3-glucan, arabinomannan,lipopolysaccharide, OK-432 (picibanil), PSK (krestin) and lentinan;metabolic antagonists such as methotrexate and fluorouracil; andantibiotics, antibiotics such as doxorubicin and mitomycin C are veryadvantageous because such combined uses extremely enhance theantioncotic effect of LK 2.

Specifically, it has been elucidated that LK 2 enhances the antioncoticeffect of chemotherapeutics.

The antioncotic effect-enhancing property of LK 2 will be explainedhereinafter.

EXPERIMENT C-1 Enhancement of antioncotic effect of chemotherapeutics byLK 2

The enhancement of antioncotic effect of chemotherapeutics by LK 2 wasstudied with malignant tumor cells.

One human malignant tumor cell suspension (10⁶ cells) was inoculated on1 ml of conventional-type nutrient culture medium supplemented withfoetal calf serum, and then cultured for one day. Thereafter, there wasadded to the culture 0.1 ml of saline containing 100 units of an LK 2specimen, prepared by the method in Experiment A-3, and/or achemotherapeutic, and cultured for an additional two days at 37° C.

                                      TABLE V                                     __________________________________________________________________________                    None                                                                              ACNU                                                                              5-FU ADM  MMC  VCR                                    Cell line       +   - + -  + -  + -  + -  +                                   __________________________________________________________________________    Hep #2          52  4 67                                                                              8  80                                                                              13 70                                                                              10 72                                                                              5  66                                  (Larynx epidermoid carcinoma)                                                 PC-8            62  2 71                                                                              10 85                                                                              6  71                                                                              5  68                                                                              8  72                                  (Lung carcinoma)                                                              MKN 7           62  4 73                                                                              6  77                                                                              5  69                                                                              3  73                                                                              6  70                                  (Gastric cancer)                                                              HLE             57  5 75                                                                              8  81                                                                              7  73                                                                              12 74                                                                              10 69                                  (Liver carcinoma)                                                             HeLa            51  5 71                                                                              12 69                                                                              7  62                                                                              7  61                                                                              10 63                                  (Cervix epitheloid carcinoma)                                                 __________________________________________________________________________

As the control, an LK 2- and chemotherapeutic-free saline was used.

After completion of the culture, the number of viable cells was countedin accordance with the method in Experiment B-1, and the growthinhibition (%) was then determined.

The concentrations of the tested chemotherapeutics were as follows:nimustine hydrochloride (ACNU), 1.0×10⁻⁶ g/ml culture; fluorouracil(5-FU), 1.5×10⁻⁸ g/ml culture; doxorubicin (ADM), 1.0×10⁻¹⁰ g/mlculture; mitomycin C (MMC), 2.5×10⁻⁹ g/ml culture; and vincristinesulfate (VCR), 1.5×10⁻¹⁰ g/culture.

The results are given in Table V.

This evidence clearly shows that LK 2 enhances extremely the antioncoticeffect of chemotherapeutics.

EXPERIMENT C-2

A group of BALB/c nude mice was transplanted subcutaneously in theirdorsum areas with small fragments of human breast cancer tissue.

After the tumor masses grew to about 200 mm³ in the bodies of theanimals, saline containing an LK 2 preparation, obtained by the methoddescribed in Experiment A-3, and/or mitomycin C (MMC) was injectedintravenously once every day for twenty days.

Thereafter, the animals were sacrificed, and the resultant tumor masseswere weighed.

As the control, an LK 2- and chemotherapeutic-free saline was used.

The results are given in Table VI.

                  TABLE VI                                                        ______________________________________                                                                 Tumor mass                                           Treatment    Dosage per day                                                                            (g)                                                  ______________________________________                                        Control      0           10.8 ± 1.0                                        LK 2         100 units/kg                                                                              6.4 ± 0.5*                                        MMC          1 mg/kg     5.1 ± 0.4*                                        LK 2         100 units/kg                                                                              4.8 ± 0.4*                                        plus MMC     0.1 mg/kg                                                        ______________________________________                                         Note: *means the values were statistically significant against the contro     in a level of significance of 5%.                                        

This evidence clearly shows that LK 2 enhances extremely the antioncoticeffect of chemotherapeutics even in vivo.

The aforementioned evidence confirms that a combination of achemotherapeutic with LK 2 shows a high antioncotic effect as comparedto the use of the chemotherapeutic alone at a low concentration to givean insufficient antioncotic effect.

The use of LK 2 as the enhancer for chemotherapeutics extremely reducesthe practical chemotherapeutic concentration, prevents the elicitationof side-effects, and widens extremely the tumor spectra of thechemotherapeutics.

The enhancer containing LK 2 according to the present invention exhibitsthe antioncotic effect-enhancing activity when used together withchemotherapeutics.

The enhancer is premixed with a chemotherapeutic, and the resultantproduct is administered simultaneously with in a single route.Alternatively, either the enhancer or a chemotherapeutic is firstadministered, then the remainder is administered with in the same ordifferent route.

The following Examples A, B and C illustrate LK 2 production,pharmaceutical compositions containing LK 2, and the LK 2-specificmonoclonal antibody, respectively.

EXAMPLE A-1

Ball-1, a human lymphoblastoid line, was inoculated on Eagle's minimalessential medium (pH 7.4), supplemented with 20% foetal calf serum, andcultured in vitro in suspension at 37° C. in the usual way. Theproliferated human cells were then washed with serum-free Eagle'sminimal essential medium (pH 7.4), and resuspended in a freshpreparation of the same culture medium to give a cell density of about1×10⁷ cells/ml. To the cell suspension was added Sendai virus in adosage of about 1,000 haemagglutination titers/ml, and the culture wasincubated at 38° C. for one day to induce LK 2 production. Aftercentrifuging the resultant culture at about 1,000×g and about 4° C., thesupernatant was dialyzed against saline containing 0.01M phosphatebuffer (pH 7.2) for 15 hours, and treated with a membrane filter. Thefiltrate was then passed through a column of anti-HuIFN antibodysimilarly as in Experiment A-1, and the unadsorbed fraction was purifiedsimilarly as in Experiment A-3 by means of affinity chromatography usinga column of an anti-LK 2 antibody-bound gel, and concentrated to obtaina concentrate with a specific LK 2 activity of about 10⁹ units/mgprotein.

The yield was about 1.5×10⁶ units/liter of the induced cell suspension.

EXAMPLE A-2

Newborn hamsters were injected with about 4 mg/hamster of conventionalantiserum prepared from rabbit to weaken their possible immunoreactionupon herero-transplantation, transplanted subcutaneously with about1×10⁷ cells/hamster of BALL-1 cell, a human lymphoblastoid line, and fedin usual way for three weeks. The tumor masses, about 15 g each, formedsubcutaneously in the animals, were extracted, minced, and disaggregatedin saline. After washing with serum-free RPMI 1640 medium (pH 7.2), theproliferated cells were resuspended in a fresh preparation of the sameculture medium to give a cell density of about 5×10⁶ cells/ml. To thecell suspension was added Sendai virus and E. coli endotoxin inrespective dosages of about 1,000 haemagglutination titers/ml and about10 μg/ml, and the culture was incubated at 37° C. for one day to induceLK 2 production. After centrifuging the culture at about 1,000×g and 4°C. to remove the sediment, the supernatant was dialyzed against salinecontaining 0.01M phosphate buffer (pH 7.2) for 21 hours, and treatedwith a membrane filter. The filtrate was purified with a column ofantibody similarly as in Example A-1, and the eluate solution wasconcentrated and lyophilized to obtain a pulverulent product with aspecific LK 2 activity of about 10⁹ units/mg protein.

The yield was about 3.2×10⁷ units.

EXAMPLE A-3

Adult nude mice were transplanted intraperitioneally with TALL-1, ahuman lymphoblastoid line, fed in usual way for five weeks, injectedintraperitoneally with Newcastle disease virus (about 3,000haemagglutination titers per nude mouse) which had been substantiallypreinactivated with uv-irradiation, and sacrificed 24 hours after theinjection, followed by harvest of their ascite fluids. The ascite fluidswere purified, concentrated, and lyophilized similarly as in Example A-2to obtain a pulverulent product with LK 2 activity.

The yield was about 3.5×10⁶ units per nude mouse.

EXAMPLE A-4

Adult mice were irradiated with about 400 rem of x-ray to weaken theirimmunoreaction, transplanted subcutaneously with Mono-1, a humanlymphoblastoid line, and fed in the usual manner for three weeks. Thetumor masses, about 10 g each, formed subcutaneously in the animals,were extracted and disaggregated similarly as in Example A-2. The humancells thus obtained were suspended similarly as in Example A-2, afterwhich Sendai virus and concanavalin A were added to the resultant cellsuspension in respective dosages of about 500 haemagglutinationtiters/ml and 0.8 μg/ml, and the culture was incubated at 37° C. for oneday to induce LK 2 production. Thereafter, the culture was purified,concentrated, and lyophilized similarly as in Example A-2 to obtain apulverulent product with LK 2 activity.

The yield was about 2.0×10⁷ units per mouse.

EXAMPLE A-5

Newborn hamsters were transplanted with Namalwa (ATCC CRL 1432), a humanlymphoblastoid line, similarly as in Example A-2, and fed in the usualway for four weeks. The tumor masses, about 20 g each, formedsubcutaneously in the animals, were extracted and disaggregated toobtain a cell suspension having a cell density of about 3×10⁶ cells/ml.To the cell suspension was added Sendai virus in a dosage of about 1,000haemagglutination titers/ml, and the culture was incubated at 36° C. fortwo days to induce LK 2 production. The culture was purified andconcentrated similarly as in Example A-1 to obtain a concentrate with LK2 activity.

The yield was about 2.2×10⁷ units per hamster.

EXAMPLE A-6

NALL-1, a human lymphoblastoid line, was suspended in saline, and placedin an about 10 ml cylindrical plastic diffusion chamber equipped with amembrane filter having a nominal pore size of about 0.5 μ. The chamberwas embedded intraperitoneally in an adult rat, and the animal was fedin the usual way for four weeks. After removal of the chamber, it wasfound that the cell density in the chamber was about 5×10⁸ cells/ml,which was at least about 10² -fold higher in comparison with the case ofproliferating in vitro in a CO₂ incubator using a nutrient culturemedium. The human cells were suspended in culture medium similarly as inExample A-2, Newcastle disease virus (about 500 haemagglutinationtiters/ml), which had been preinactivated with uv-irradiation, andphytohaemagglutinin (about 50 μg/ml), were added and the culture wasincubated at 37° C. for one day to induce LK 2 production. Thereafter,the culture was purified, concentrated, and lyophilized similarly as inExample A-2 to obtain a pulverulent product with LK 2 activity.

The yield was about 8×10⁶ units per rat.

EXAMPLE A-7

CCRF-CEM (ATCC CCL 119), a human lymphoblastoid line was inoculated inthe allantoic cavities of embrynated eggs which had been incubated at37° C. for five days, and the eggs were further incubated at thistemperature for an additional one week. The proliferated human cellswere harvested from the eggs, and suspended similarly as in Example A-1to give a cell density of 5×10⁶ cells/ml. To the cell suspension wasthen added Sendai virus (about 500 haemagglutination titers/ml), and theculture was incubated at 37° C. for one day to induce LK 2 production.The resultant culture was purified and concentrated similarly as inExample A-2 to obtain a pulverulent product with LK 2 activity.

The yield was about 7.0×10⁵ units per ten embryonated eggs.

EXAMPLE B-1 Injection

Five hundred thousand units of an LK 2 specimen, prepared by the methodin Example A-2, were dissolved in 200 ml saline, and filtered understerile conditions by use of a membrane filter. Two ml aliquots of thefiltrate were distributed into sterilized glass vials, lyophilized, andsealed to obtain a pulverulent injection.

The injection is favorably usable alone or as an enhancer in combinationwith a chemotherapeutic such as melphalan, methotrexate or doxorubicinfor treating breast cancer, lung carcinoma, liver carcinoma andleukaemia.

EXAMPLE B-2 Injection

A pulverulent injection was prepared similarly as in Example B-1, exceptthat 3×10⁸ units of HuIFN-α derived from a human lymphoblastoid cellwere dissolved in 200 ml of saline together with 5×10⁵ units of LK 2.

The injection is favorably usable alone or as an enhancer in combinationwith a chemotherapeutic such as tegafur, mitomycin C or vincristinesulfate for treating breast cancer, lung carcinoma, liver carcinoma, andleukaemia, and its therapeutic efficacy is superior to that of theinjection at Example B-1.

EXAMPLE B-3 Ointment

An LK 2 specimen, prepared by the method in Example A-3, was kneadedwith a minimal amount of liquid paraffin to homogeneity. To the mixturewas then added white petrolatum in the usual way to obtain an ointmentwith an LK 2 content of 20,000 units/g.

The ointment is favorably usable alone or as an enhancer in combinationwith a chemotherapeutic such as cyclophosphamide, fluorouracil orvincristine sulfate for treating skin carcinoma, breast cancer andlymphoma.

EXAMPLE B-4 Collyrium

A mixture of 800 ml distilled water, 5 ml β-phenylethyl alcohol and20,000,000 units of an LK 2 specimen, prepared by the method in ExampleA-4, was admixed with sodium chloride in an additional amount ofdistilled water to obtain 1,000 ml of an isotonic solution.

The solution is favorably usable alone or as an enhancer in combinationwith a chemotherapeutic such as cytarabine or vincristine sulfate fortreating retinoblastoma.

EXAMPLE B-5 Enteric coated tablet

Enteric coated tablets were prepared according to the conventionalmethod by tabletting a mixture of starch, maltose, and an LK 2 specimenprepared by the method in Example A-7 to give an LK 2 content of 200,000units per tablet (100 mg), followed by coating the tablets withphthalate ester of methyl cellulose.

The tablets are favorably usable alone or as an enhancer in combinationwith a chemotherapeutic such as doxorubicin, fluorouracil or mitomycin Cfor treating colon carcinoma and liver carcinoma.

EXAMPLE C-1

Mice were immunized similarly as in Experiment A-2, except that ahigh-purity LK 2 obtained by the method described in Experiment A-3 wasused as the antigen. Thereafter, the spleen cells of the animals weresuspended together with a mouse myeloma line,P3-NS-1/1-Ag4-1(ATCCT1B18), a product of Dainippon Pharmaceutical Co.,Ltd., Osaka, Japan, in a salt solution containing 140 mm NACL, 54 mmKCL, 1 mm NaH₂ PO₄, and 2 mM CaCl₂ to give a respective cell density of10⁴ cells/ml. To the cell suspension was added a fresh salt solution ofthe same composition but containing, in addition, Sendai virus, whichhad been inactivated with uv-irradiation, under ice-chilled conditions,and the mixture was allowed to stand for five minutes. Thereafter, themixture was diluted about 20-times in 37° C. RPMI 1640 medium, and thehybridoma cell capable of producing anti-LK 2 antibody were clonedsimilarly as in Experiment A-2. The cloned hybridoma cells weretransplanted intraperitoneally into 7-day old hamsters (about 10⁷ cellsper hamster) whose immunoreaction had been weakened with a conventionalprocedure, and the monoclonal anitbody was recovered from the bodies ofthe animals similarly as in Experiment A-2.

Like the monoclonal antibody prepared in Experiment A-2, the productexhibited an immunologically-specific neutralization with the cytotoxicactivity of LK 2.

The stability of the monoclonal antibody in aqueous solution was studiedby assaying the residual neutralizing activities after incubating underprescribed conditions: On incubation at pH 7.2 and differenttemperatures for 30 minutes, 80% or more of the activity was retained at60° C., but 90% or more was lost at 70° C. After incubation at 4° C. anddifferent pH levels for 16 hours, the activity was stable in the pHrange of 2.0-11.0.

On studying several properties of the monoclonal antibody, it was foundthat the present monoclonal antibody was resistant to 2-mercaptoethanol,and effects a specific antigen-antibody reaction with anti-mouseimmunoglobulin G antibody. Thus, the present monoclonal antibody isgrouped into the class of immunoglobulin G antibody.

EXAMPLE C-2

A monoclonal anti-LK 2 antibody was prepared similarly as in ExampleC-1, except that a mouse myeloma line, SP2/0-Ag14 (ATCC CRL 1581),available from Dainippon Pharmaceutical Co., Ltd., Osaka, Japan, wasreplaced for P3-NS-1/1-Ag4-1.

On studying the immunologically-specific neutralization by themonoclonal antibody with the cytotoxic activity of LK 2, a resultsimilar to that obtained in Example C-1, was obtained. Thus, themonoclonal antibody is also grouped into the class of immunoglobulin Gantibody.

While the invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that the foregoing and other changes can bemade therein without departing from the spirit and scope of theinvention.

We claim:
 1. A process to prepare a lymphokine (LK 2) having thefollowing physicochemical properties:(1) Molecular weight: 20,000±2,000daltons; (2) Isoelectric point: pI=6.2±0.3; (3) Electrophoreticmobility: on Disc-PAGE, Rf=0.29±0.02; (4) uv-Absorption spectrum: anabsorption maximum at a wave length of about 280 nm; (5) Solubility insolvents: soluble in water, saline and phosphate buffer, scarcelysoluble or insoluble in ethyl ether, ethyl acetate or chloroform; (6)Coloring reaction: protein-positive by Lowry's method or the microburetmethod, saccharide-positive by the phenol-sulfuric acid method oranthrone-sulfuric acid method; (7) Biological activities: cytotoxic on L929 cell, substantially not growth-inhibitive on KB cell, substantiallyfree of interferon activity; (8) Stability in aqueous solution: stableup to 60° C. when incubated at pH 7.2 for 30 minutes, stable in the pHrange of 4.0-11.0 when incubated at 4° C. for 16 hours; and (9)Stability on cryopreservation: stable at -10° C. over a period of onemonth or longer,said process comprising the steps of: exposing humancells capable of producing LK 2 to an LK 2 inducer; and separating aproduct consisting essentially of the accumulated LK 2 from the productof said exposing step.
 2. The process in accordance with claim 1,wherein said human cells capable of producing LK 2 are obtained byproliferating a human cell capable of producing LK 2, and wherein saidexposing step takes place under conditions sufficient to accumulate asubstantial amount of LK
 2. 3. The process in accordance with claim 2,wherein the human cell capable of producing LK2 is proliferated invitro.
 4. The process in accordance with claim 2, wherein the human cellcapable of producing LK 2 is proliferated in vivo.
 5. The process inaccordance with claim 1, wherein the human cells capable of producing LK2 are obtained by:transplanting human cells capable of producing LK 2 toa non-human warm-blooded animal; feeding the animal to allow the humancells to utilize the nutrient body fluid of the animal for itsproliferation; and extracting and disaggregating the resultant tumorformed in the animal.
 6. The process in accordance with claim 5, whereinthe non-human warm-blooded animal is a fowl or a mammal.
 7. The processin accordance with claim 1, wherein the human cells capable of producingLK 2 are obtianed by:suspending human cells capable of producing LK 2 ina diffusion chamber in which the nutrient body fluid of a non-humanwarm-blooded animal is supplied to the human cells; embedding or placingsaid chamber in or on a non-human warm-blooded animal in a manner suchthat the nutrient body fluid of the animal is supplied to the humancells within said chamber; feeding the animal to allow the human cellsto utilize the nutrient body fluid for their proliferation; andcollecting the proliferated human cells from the chamber.
 8. The processin accordance with claim 7, wherein the non-human warm-blooded animal isa fowl or a mammal.
 9. The process in accordance with claim 5, whereinthe non-human warm-blooded animal is immunodeficient orimmunosuppressed.
 10. The process in accordance with claim 7, whereinsaid diffusion chamber equipped with one or more membrane filters,hollow fibers or ultra-filters having a nominal pore size of 10⁻⁷ -10⁻⁵m.
 11. The process in accordance with claim 2, wherein the human cellcapable of producing LK 2 is human leukocyte or human lymphocyte. 12.The process in accordance with claim 2, wherein the human cell capableof producing LK 2 is a lymphoblastoid cell.
 13. The process inaccordance with claim 2, wherein the human cell capable of producing LK2 is a member selected from the group consisting of Namalwa (ATCC CRL1432), BALL-1, TALL-1, NALL-1, M-7002, B-7101, JBL, EBV-Sa, EBV-Wa,EBV-HO, MOLT-3 (ATCC CRL 1552), CCRF-SB (ATCC CCL 120), CCRF-CEM (ATCCCCL 119), BALM 2, DND-41, and Mono-1.
 14. The process in accordance withclaim 2, wherein said exposing step is carried out in vitro.
 15. Theprocess in accordance with claim 2, wherein said exposing step iscarried out in vivo.
 16. The process in accordance with claim 2, whereinthe LK 2 inducer is a member selected from the group consisting ofinterferon-alpha inducer, interferon-gamma inducer, and mixturesthereof.